Platelet preservation package comprising a short to ultra-short acting antiplatelet agent and anticoagulant with an oxygen carrier

ABSTRACT

The present invention relates to compositions and methods for storing platelets to preserve the function and freshness of the platelets. More particularly, the present invention relates to the use of a preservative composition having an antiplatelet agent, an anticoagulant, and an oxygen carrier, for maintaining the freshness of platelets. Additionally, the composition may also contain an ultra-short acting broad spectrum anti-microbial agents. The preservative composition may be used to store platelets in a liquid state, a frozen state, or a freeze-dried state.

This application is a continuation application of the U.S. patentapplication Ser. No. 11/330,132, filed Jan. 12, 2006 which claimspriority from U.S. Provisional Application Ser. No. 60/643,107 filedJan. 12, 2005. The entirety of all of the aforementioned applications isincorporated herein by reference.

FIELD

The present invention relates to a composition and method for extendingthe shelf-life of platelets. More particularly, the present inventionrelates to a preservative composition comprising an ultra-short actingantiplatelet agent and/or ultra-short acting anticoagulant and an oxygencarrier. The composition is particularly useful in extending life andmaintaining the efficacy of platelets.

BACKGROUND

Whole blood contains the nutrients necessary to nourish tissues andorgans in multi-cellular organisms. Arterial blood, or, blood carried inthe arteries, is the blood in which oxygen and nutrients are transportedto tissues. Venous blood, or blood carried in the veins, is the blood inwhich carbon dioxide and metabolic by-products are transported forexcretion. These processes make it possible for nourishment to reachorgans and tissues, and to sustain life in multi-cellular organisms.

Whole blood contains three components: (a) a fluid portion (plasma) withsuspended elements, such as (b) red blood cells and white blood cells,and (c) platelets. The red blood cells are oxygen carriers and comprisean efficient oxygen carrier, called hemoglobin. Plasma is the liquidportion of the whole blood and contains nutrients, electrolytes(dissolved salts), gases, albumin, clotting factors, wastes, andhormones.

When blood vessels are damaged, cell fragments released from the bonemarrow, called platelets, adhere to the walls of blood vessels and formclots to prevent blood loss. It is important to have adequate numbers ofnormally functioning platelets to maintain effective clotting, orcoagulation, of the blood. Occasionally, when the body undergoes trauma,or when the platelets are unable to function properly, it is necessaryto replace or transfer platelet components of blood into a patient. Mostcommonly, platelets are obtained from volunteer donors either as acomponent of a whole blood unit, or via plateletpheresis (withdrawingonly platelets from a donor and re-infusing the remaining of the bloodback into the donor). The platelets then are transferred to a patient asneeded, a process referred to as “platelet transfusion”.

Platelet transfusion is indicated under several different scenarios. Forexample, an acute blood loss, either during an operation or as a resultof trauma, can cause the loss of a large amount of platelets in a shortperiod of time. Platelet transfusion is necessary to restore a normalability to control blood flow, or haemostasis. In a medical setting, anindividual can develop a condition of decreased number of platelets, acondition known as thrombocytopenia. The condition can occur as a resultof chemotherapy, and requires platelet transfusion to restore normalblood clotting.

Unlike red blood cells, which can be stored for forty-five (45) days,platelets can be stored for only five to seven days. The short storageterm, or shelf-life, of the platelets severely limits the useful spanfor a platelet supply. A consequence of this short shelf-life is thatplatelets must be collected close to their time of use, which makes itextremely difficult to coordinate platelet collection and plateletsupply.

One reason platelets have such short shelf-life is that platelets becomeactivated during the process of collection. The activation process leadsto externalization of platelet canalicular surfaces exposing receptorsites, such as GPIIb/IIIa. Phosphatidylserine residues on activatedplatelets tend to cause platelet aggregation, which results in celldeath (i.e., apoptosis) upon re-infusion into patients. Thus, a plateletfunctional half-life is significantly reduced.

Another reason that platelets have a short shelf-life is that aninadequate oxygen supply alters the metabolic activity of the platelets.In an environment lacking a sufficient oxygen supply, the plateletsundergo an anaerobic mechanism leading to accumulation of lactic acid.The increased concentration of lactic acid causes a drop in pH, andresults in cell death. Although platelets can be stored in gas permeablebags using a shaker bath under a stream of air to help overcome thisproblem, such storage methods are costly and extremely inefficient andinadequate in meeting the oxygen requirements of the stored platelets.

Platelet sterility is difficult to maintain because platelets cannot bestored at low temperatures, for example 4° C. to 5° C. As previouslymentioned, a low storage temperature for the platelets initiates anactivation process within the platelets that leads to aggregation andcell death. Unfortunately, bacterial growth in the platelet medium atsuitable storage temperatures, e.g., room temperature, can lead to anunacceptable occurrence of bacterial contamination in platelets used fortransfusion. As a result, the Food and Drug Administration (FDA) limitsthe storage time of platelets to five (5) days, thereby safeguarding thetransfusion supply from bacterial contamination.

Many sterilization methods have been suggested. Platelet compositionstypically can be sterilized by radiation, chemical sterilization, or acombination thereof. For example, a method of inactivating viral andbacterial blood contaminants using a quinoline as a photosensitizer isdisclosed in U.S. Pat. No. 5,798,238. Other classes of photosensitizersare, for example, psoralens, coumarins, or other polycyclic ringcompounds, as disclosed in U.S. Pat. No. 5,869,701; quinolones, asdisclosed in U.S. Pat. No. 5,955,256; free radical and reactive forms ofoxygen, as disclosed in U.S. Pat. Nos. 5,981,163 and 6,087,141; andphenothiazin-5-ium dyes, as disclosed in U.S. Pat. No. 6,030,767. U.S.Pat. No. 6,106,773 discloses another method for disinfecting biologicalfluids, including platelets, by contacting the biological fluids with aniodinated matrix material.

These sterilization methods, however, do not extend storage life but onthe contrary, appear to significantly decrease their functionality. Toeffectively extend the shelf life of platelets, not only aresterilization methods for preventing contamination of the plateletsimportant, but it also would be beneficial to provide improved methodsto protect the platelets during the sterilization. It would also bebeneficial to provide a convenient, effective preservative solution forprolonging the shelf-life of the platelets, while maintaining thefunctionality and freshness of the platelets. In addition, it would bebeneficial to provide a method or composition for storing platelets thatrequires less management of the surrounding platelet storageenvironment.

SUMMARY

One aspect of the present invention relates to a platelet preservationcomposition comprising (a) an antiplatelet agent, (b) an anticoagulant,and (c) an oxygen carrier. The preservative composition, when added tofreshly collected platelets, permits an extended storage of platelets atambient temperatures, and maintains blood clotting properties withoutaffecting the half-life of the platelets in circulation aftertransfusion.

In one embodiment, the antiplatelet agent is capable of reversiblyblocking platelet activation and/or aggregation by blocking sites on theplatelet surface.

In another embodiment, the antiplatelet agent is selected from the groupconsisting of a compound that binds to, or associates with a GPIIb/IIIareceptor site, a non-steroidal anti-inflammatory drug (NSAID), a calciumchannel blocker, α-blocker, β-adrenergic blocker, and mixtures thereof.

In another embodiment, the anticoagulant is a compound that reversiblyinhibits factor Xa, or factor IIa, or both.

In another embodiment, the anticoagulant is a short-to-ultrashort actingXa inhibitor.

In another embodiment, the anticoagulant is a short-to-ultrashort actingIIa inhibitor.

In another embodiment, the oxygen carrier is a hemoglobin-based oxygencarrier.

In yet another embodiment, the oxygen carrier is selected from the groupconsisting of hemoglobin, ferroprotoporphyrin, perfluorochemicals, andmixtures thereof.

In yet another embodiment, each the hemoglobin-based oxygen carrier issubstantially free of red cell membrane contaminants.

In another embodiment, the platelet preservation composition furthercomprises a short or ultra-short acting anti-microbial agent.

In another embodiment the platelet preservation composition can be usedin a concentration range from about 0.001 to about 5 mg in any settingthat requires the circulation of blood outside the body such as inpatients undergoing open heart surgery, renal dialysis, plasmapheresis,and other procedures requiring platelet supplementation.

Another aspect of the invention relates to a preserved plateletcomposition comprising the platelet preservation composition describedabove and inactivated, functional platelets.

In one embodiment, the platelets in the preserved platelet compositionare substantially free of red blood cells or other blood nutrients.

Another aspect of the present invention relates to a method of extendingthe shelf-life of platelets. The method comprises the steps of (a)admixing a preservative composition with inactivated, functionalplatelets and (b) store the preserved platelet in an oxygen-permeablecontainer or an oxygen-impermeable container, wherein the preservativecomposition comprises a pharmaceutically acceptable antiplatelet agent,a pharmaceutically acceptable anticoagulant, and a pharmaceuticallyacceptable oxygen carrier.

In an embodiment, the inactivated, functional platelets are freshlycollected platelets and are substantially free of red blood cells andother blood nutrients.

In another embodiment, the preservative composition further comprises ashort-to-ultrashort acting broad spectrum anti-microbial agent.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are the diagrams of TEG and tracing showing theinitiation and completion phases of platelet-fibrin clot initiated by TF(25 ng) under shear in human whole blood (WB).

FIG. 2A shows the initiation of platelet/fibrin clot by tissue factor(TF) in human blood using thromboelastography (TEG). FIG. 2B shows theinhibition of platelet/fibrin clot induced by TF with differentconcentrations of c7E3 (antibody against platelet GPIIb/IIIa). FIG. 2Cshow comparable effect of c7E3 on platelet aggregation andplatelet/fibrin clot.

FIG. 3 is a diagram showing a protocol of GP IIb/IIIa antagonists andplatelet fibrinogen binding short to ultra-short acting GP IIb/IIIaantagonists including those that are separated from platelet upon sizeexclusion chromatography.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description is presented to enable any personskilled in the art to make and use the invention. For purposes ofexplanation, specific nomenclature is set forth to provide a thoroughunderstanding of the present invention. However, it will be apparent toone skilled in the art that these specific details are not required topractice the invention. Descriptions of specific applications areprovided only as representative examples. Various modifications to thepreferred embodiments will be readily apparent to one skilled in theart, and the general principles defined herein may be applied to otherembodiments and applications without departing from the scope of theinvention. The present invention is not intended to be limited to theembodiments shown, but is to be accorded the widest possible scopeconsistent with the principles and features disclosed herein.

One aspect of the present invention relates to a preservativecomposition that preserves the freshness of platelets. The preservativecomposition extends the shelf-life of donated platelets. Thepreservative composition comprises an antiplatelet agent, ananticoagulant, and an oxygen carrier.

The term “pharmaceutically acceptable” as used herein refers to asubstance that complies with the regulations enforced by the FDAregarding the safety of use in a human or animal subject or a substancethat has passed the FDA human safety trials. The term “pharmaceuticallyacceptable antiplatelet agent”, for example, refers to an active agentthat prevents, inhibits, or suppresses platelet adherence and/oraggregation, and comports with guidelines for pharmaceutical use as setforth by the FDA.

Any agent that reversibly impedes platelet activation and/or aggregationby blocking sites on the platelet surface can be used as theantiplatelet agent in the present invention. As used herein, the term“reversible” or “reversibly” refers to an act, such as binding orassociating, that is capable of reverting back to an original conditionprior to the act, for example the state of being unbound ordisassociated, either with or without the assistance of an additionalconstituent.

As used herein, the term “effective amount” refers to a quantity that iscapable of achieving an intended effect.

The temperature used in the present invention is from about −80° C. to42° C. As used herein, the term “room temperature” or “ambienttemperature” refers to a temperature in the range of 12° C. to 30° C.;the term “body temperature” refers to a temperature in the range of 35°C. to 42° C.; the term “refrigeration temperature” refers to atemperature in the range of 0° C. to 12° C.; and the term “freezingtemperature” refers to a temperature below 0° C.

Generally, agents that can impede platelet activation and/or aggregationinclude, but are not limited to, heparin, heparin substitutes,prothrombopenic anticoagulants, platelet phosphodiesterase inhibitors,dextrans, and the like, or mixtures thereof. Examples of heparin andheparin substitutes include, but are not limited to, heparin calcium,such as calciparin; heparin low-molecular weight, such as enoxaparin andlovenox; heparin sodium, such as heparin, lipo-hepin, liquaemin sodium,and panheprin; and heparin sodium dihydroergotamine mesylate. Suitableprothrombopenic anticoagulants are, for example, anisindione, dicumarol,warfarin sodium, and the like. More specific examples ofphosphodiesterase inhibitors suitable for use in the invention include,but are not limited to, anagrelide, dipyridamole, pentoxifyllin, andtheophylline. Examples of dextrans are, for example, dextran 70, such asHYSKON® (CooperSurgical, Inc., Shelton, Conn., U.S.A.) and MACRODEX®(Pharmalink, Inc., Upplands Väsby, Sweden), and dextran 75, such asGENTRAN® 75 (Baxter Healthcare Corporation, Deerfield, Ill., U.S.A.).

Antiplatelet agents can include, but are not limited to, active agentsthat bind GPIIb/IIIa sites in a reversible manner and non-steroidalanti-inflammatory drugs (NSAIDs). In a preferred composition, the activeagents for binding to or associating with GPIIb/IIIa sites have acirculating half-life of inhibition of 4 hours or less. Examples ofsuitable antiplatelet agents for binding GPIIb/IIIa sites in areversible manner are eptifibatide (INTEGRILIN®, Schering-PloughCorporation, Kenilworth, N.J., U.S.A.), orbofiban, xemilofiban,Lamifiban, tirofiban, abciximab, XJ757, DUP728, XR299, linear or novelcyclic RGD peptide analogs, cyclic peptides, peptidomimetics andnon-peptide analogs conjugated to Nitric Oxide donor and the like, andmixtures thereof.

Non-steroidal anti-inflammatory drugs (NSAIDS) are commonly available,and typically are used for treating inflammation. Generally, NSAIDS canhave a salicylate-like or non-salicylate structure. NSAIDS suitable forthe invention can be salicylate-like or non-salicylate NSAIDS that bindreversibly and inhibit platelet aggregation in vitro, but are clearedrapidly, i.e. quickly eliminated from the body, when infused (typically,in less than about 2 hours). NSAIDS suitable for the invention include,but are not limited to, for example, salicylate-like NSAIDS, such asacetaminophen, carprofen, choline salicylate, magnesium salicylate,salicylamide, sodium salicylate, sodium thiosulfate, and the like, andmixtures thereof. Examples of non-salicylate NSAIDS include, but are notlimited to, diclofenac sodium, diflunisal, etodolac, fenoprofen calcium,flurbiprofen, hydroxychloroquin, ibuprofen, indomethacin, ketoprofen,ketorolac tromethamine, meclofenamate sodium, mefenamic acid,nabumetone, naproxen, naproxen sodium, oxyphenbutazone, phenylbutazone,piroxicam, sulfinpyrazone, sulindac, tolmetin sodium, dimethylsulfoxide, and the like, and mixtures thereof.

In addition, any agent that inhibits chemical pathways within theplatelets leading to reduction in platelet activation, is suitable forthe invention. Typically, agents that inhibit chemical pathways leadingto reduced platelet activation are calcium sequestering agents, such ascalcium channel blockers, α-blockers, β-adrenergic blockers, and thelike, and mixtures thereof. More specific examples of calciumsequestering agents include, but are not limited to, anticoagulantcitrate dextrose solution, anticoagulant citrate dextrose solutionmodified, anticoagulant citrate phosphate dextrose solution,anticoagulant sodium citrate solution, anticoagulant citrate phosphatedextrose adenine solution, potassium oxalate, sodium citrate, sodiumoxalate, amlodipine, bepridil hydrochloride, diltiazem hydrochloride,felodipine, isradipine, nicardipine hydrochloride, nifedipine,nimodipine, verapamil hydrochloride, doxazosin mesylate,phenoxybenzamine hydrochloride, phentolamine mesylate, prazosinhydrochloride, terazosin hydrochloride, tolazoline hydrochloride,acebutolol hydrochloride, atenolol, betaxolol hydrochloride, bisoprololfumarate, carteolol hydrochloride, esmolol hydrochloride, indoraminehydrochloride, labetalol hydrochloride, levobunolol hydrochloride,metipranolol hydrochloride, metoprolol tartrate, nadolol, penbutololsulfate, pindolol, propranolol hydrochloride, terazosin hydrochloride,timolol maleate, guanadrel sulfate, guanethidine monosulfate,metyrosine, reserpine, and the like, and mixtures thereof.

The antiplatelet agent can be used in conjunction with apharmaceutically acceptable oxygen carrier. The oxygen carrier can beany suitable red blood cell substitute. Typically, the oxygen carrier isan acellular hemoglobin-based oxygen carrier substantially free of redcell membrane (stroma) contaminants. The term “pharmaceuticallyacceptable oxygen carrier” as used herein refers to a substance that haspassed the FDA human safety trials at a hemoglobin dosage of 0.5 g/kgbody weight or higher. An oxygen carrier suitable for the invention canbe hemoglobin, ferroprotoporphyrin, perfluorochemicals (PFCs), and thelike. The hemoglobin can be from human or any other suitable mammaliansource. In a preferred embodiment, the preservative composition has ahemoglobin concentration from the range of 1 to 18 gm/dl and amethemoglobin concentration of less than about 5%. The hemoglobin basedoxygen carrier will be chemically modified to mimic the oxygen loadingand unloading characteristics of fresh red blood cells. Additionally,the chemical modification will enhance the buffering capacity of thepreferred embodiment and preserve normal physiologic pH.

The amount of the antiplatelet agent present in the preservativecomposition depends on the type of antiplatelet agent. The amount ofantiplatelet agent is sufficient to reversibly inhibit binding to aligand or site on the platelet in a manner that is sufficient to inhibitplatelet function, when bound. For GPIIb/IIIa inhibitors, suitableamounts in the preservative composition are about 0.5 mg to about 3 mgfor 50 ml of acellular hemoglobin-based oxygen carrier substantiallyfree of red cell membrane (stroma) contaminants. NSAIDs, for example,ibuprofen, are present in the preservative composition in an amount ofabout 20 mg to about 60 mg for each 50 ml of acellular hemoglobin-basedoxygen carrier that is substantially free of red cell membranecontaminants.

The anticoagulants of the present invention include Xa inhibitors, IIainhibitors, and mixtures thereof. Various direct Xa inhibitors weresynthesized and advanced to clinical development (Phase I-II) for theprevention and treatment of venous thromboembolic disorders and certainsettings of arterial thrombosis [Hirsh and Weitz, Lancet, 93:203-241,(1999); Nagahara et al., Drugs of the Future, 20: 564-566, (1995); Pintoet al., 44: 566-578, (2001); Pruitt et al., Biorg. Med. Chem. Lett., 10:685-689, (2000); Quan et al., J. Med. Chem. 42: 2752-2759, (1999); Satoet al., Eur. J. Pharmacol., 347: 231-236, (1998); Wong et al, J.Pharmacol. Exp. Therapy, 292:351-357, (2000)]. A direct anti-IIa(thrombin) such as melagatran, the active form of pro-drug ximelagatran[Hirsh and Weitz, Lancet, 93:203-241, (1999); Fareed et al., CurrentOpinion in Cardiovascular, pulmonary and renal investigational drugs,1:40-55, (1999)]. Additionally, a number of VIIa inhibitors andanti-tissue factors are in pre-clinical and early stage of clinicaldevelopment. Formulation of zwitterionic short acting GPIIb/IIIaantagonists with small molecules direct Xa inhibitor, IIa inhibitor ormixed Xa and IIa inhibitors as defined by Mousa et al., Athero. Thromb.Vasc. Biol., 2000) would provide an optimal platelet preservation. Shortto ultra-short acting GPIIb/IIIa antagonist might include eptifibatide,tirofiban, DUP728, abciximab (Reopro), lefradafiban, sibrafiban,orbofiban, xemilofiban, lotrafiban, XJ757, and XR299 (Class II). Shortto ultra-short acting anticoagulant might include those direct Xainhibitors, such as DX-9065a, RPR-120844, BX-807834 and SEL series Xainhibitors, or direct IIa inhibitors, such as DUP714, hirulog, hirudin,and other short acting peptidomimetic or non-peptide Xa inhibitors, IIainhibitors, or mixtures thereof. Some of these inhibitors are discussedin more details below.

DX-9065a is a synthetic, non-peptide, propanoic acid derivative, 571 Da,selective factor Xa inhibitor (Daiichi). It directly inhibits factor Xain a competitive manner with an inhibition constant in the nanomolarrange [Herbert et al., J. Pharmacol. Exp. Ther. 276:1030-1038 (1996);Nagahara et al., Eur. J. Med. Chem. 30(suppl):140s-143s (1995)].

As a non-peptide, synthetic factor Xa inhibitor, RPR-120844(Rhone-Poulenc Rorer), is one of a series of novel inhibitors whichincorporate 3-(S)-amino-2-pyrrolidinone as a central template [Ewing etal., Drugs of Future 24(7):771-787 (1999)]. This compound has a Ki of 7nM with selectivity >150-fold over thrombin, activated protein C,plasmin and t-PA. It prolongs the PT and ∥ ┐PTT in aconcentration-dependent manner, being more sensitive to the ┌ ∥PTT. Itis a fast binding, reversible and competitive inhibitor of factor Xa.

BX-807834 has a molecular weight of 527 Da and a Ki of 110 pM for factorXa as compared to 180 pM for TAP and 40 nM for DX-9065a [Baum et al.,Circulation. 98 (17), Suppl 1: 179, (1998)].

SEL Series Xa Inhibitors

The SEL series of novel factor Xa inhibitors (SEL-1915, SEL-2219,SEL-2489, SEL-2711: Selectide) are pentapeptides based on L-amino acidsproduced by combinatorial chemistry. They are highly selective forfactor Xa and potency in the pM range. The Ki for SEL 2711, one of themost potent analogues, is 0.003 M for factor Xa and 40 M for thrombin[Ostrem et al., Thromb. Haemost. 73:1306 (1995); Al-Obeidi and Ostrem.,Exp. Opin. Ther. Patents 9(7):931-953 (1999)].

The preservative composition of the present invention may furthercomprise a short to ultra-short acting broad spectrum anti-microbialagent. Examples of such an agent include, but are not limited to, theagents listed below:

1. Penicillin—a group of antibiotics produced either by Penicillium(natural penicillins) or by adding side chains to the β-lactam ring(semisynthetic penicillins).

2. Natural penicillins—the first agents of the penicillin family thatwere produced; ex: penicillins G and V.

3. Semisynthetic penicillins—modifications of natural penicillins byintroducing different side chains that extend the spectrum ofantimicrobial activity and avoid microbial resistance.

4. Monobactam—a synthetic antibiotic with a β-lactam ring that ismonocyclic in structure.

5. Cephalosporin—an antibiotic produced by the fungus Cephalosporiumthat inhibits the synthesis of gram-positive bacterial cell walls.

6. Carbapenems—antibiotics that contain a β-lactam antibiotic andcilastatin.

7. Vancomycin—an antibiotic that inhibits cell wall synthesis.

8. Isoniazid (INH)— a bacteriostatic agent used to treat tuberculosis.

9. Ethambutol—a synthetic antimicrobial agent that interferes with thesynthesis if RNA.

10. Aminoglycoside—an antibiotic consisting of amino sugars and anaminocyclitol ring, such as streptomycin.

11. Tetracycline—a broad-spectrum antibiotic that interferes withprotein synthesis.

12. Chloramphenicol—a broad-spectrum bacteriostatic chemical.

13. Macrolide—an antibiotic that inhibits protein synthesis, such aserythromycin.

14. Rifamycin—an antibiotic that inhibits bacterial RNA synthesis.

15. Quinolone—an antibiotic that inhibits DNA replication by interferingwith the enzyme DNA gyrase.

16. Fluoroquinolone—a synthetic antibacterial agent that inhibits DNAsynthesis.

17. Sulfonamide—a bacteriostatic compound that interferes with folicacid synthesis by competitive inhibition.

18. Synergism—the principle whereby the effectiveness of two drugs usedsimultaneously is greater than that of either drug used alone.

19. Polyene antibiotic—an antimicrobial agent that alters sterols ineucaryotic plasma membranes and contains more than four carbon atoms andat least two double bonds.

20. Imidazole—an antifungal drug that interferes with sterol synthesis.

21. Triazole—an antifungal antibiotic used to treat systemic fungalinfections.

22. Griseofulvin—a fungistatic antibiotic.

In another embodiment, the platelet preservation composition can be usedin a concentration range from about 0.001 to about 5 mg in any settingthat requires the circulation of blood outside the body such as inpatients undergoing open heart surgery, renal dialysis, plasmapheresis,and other procedures requiring platelet supplementation.

In another aspect, the present invention relates to a method forextending the shelf-life of platelets using the preservative compositiondescribed above. The method comprises admixing the preservativecomposition of the present invention with inactivated, functionalplatelets to form preserved platelets with extended shelf-life. Theinactivated, functional platelets can be in the form of whole blood, aplatelet-containing component of whole blood, or isolated plateletssubstantially free of red blood cells and other blood nutrients. Thepreserved platelets can be stored at room temperature, refrigerationtemperatures, or freezing temperatures in liquid, frozen, orfreeze-dried state to maintain the freshness and function of theplatelets.

The preservative composition can be directly added to a blood collectionbag, or be kept in a separate bag and combined with the blood aftercollection. The blood in the collection bag optionally can be treatedwith an anticoagulant. In a typical setting, the preservativecomposition is added directly to the blood collection bag.

Typically, the blood is whole blood isolated from a mammal, for use inthe same species. In the case of a human, the blood is isolated andseparated into the three core components of whole blood, i.e., plasma,cells, and platelets. The whole blood, or only the platelet component ofthe whole blood, can be treated with the preservative composition. Ifwhole blood is treated, a preferred embodiment contemplates the use ofonly some components of the proposed preservative composition, such asthe antiplatelet agent and anticoagulant, for whole blood storage. Theblood can then be fractionated and the platelet component can be furthermixed with the preservative composition of the present invention forstorage.

If the platelets will be subsequently frozen or freeze dried, theplatelets will be mixed with the preservative composition beforefreezing.

Functional activities of platelets are determined by their ability toaggregate in the presence of certain biological agents and theirmorphology. Platelet function also can be assessed by the maintenance ofthe pH upon limited storage of a solution containing the platelets andin vivo haemostatic effectiveness using the rabbit kidney injury modeldescribed in Krishnamurti et al., Transfusion, 39:967 (1999). Structuralintegrity of platelets is assessed by in vivo survival followingradiolabeling with carbon-15 or indium-111 and identification of thepresence of specific platelet antigens.

The preservative composition of the present invention is used in anamount of about 60 to about 200 ml at room temperature for about oneunit of platelets at room temperature, typically about 80 to about 100ml of platelets. Alternatively, the preservative composition of thepresent invention is combined with about one unit of whole blood,typically about one pint, and separated into various components toafford about one-sixth to about one-tenth whole blood unit of treatedplatelets. To achieve the full advantage of the present invention, thepreservative composition contains an antiplatelet agent dissolved inabout 45 to about 55 ml of an oxygen carrier. When used with an unit ofwhole blood, the antiplatelet agent can also be dissolved in about 45 toabout 55 ml of normal saline to preserve the freshness of the plateletswithout an oxygen carrier. The selection of an antiplatelet agent and anoxygen carrier, and the determination of the amounts for including suchcomponents in the preservative composition, are within the capabilityof, or can be readily determined by, those skilled in the art ofpreparing preserved platelet compositions.

The platelets used in the invention can be sterilized by chemicalsterilization, radiation, or a combination thereof, in the presence ofthe preferred embodiment. For example, the platelets can be sterilizedby chemical filtration; ultraviolet radiation, such as UVA, UVB, andUVC; gamma-radiation; ionizing radiation, such as x-ray radiation; or byusing a chemical as a photosensitizer. Methods for sterilizing plateletsare well known in the art and include, for example, the methodsdescribed in U.S. Pat. Nos. 5,798,238; 5,869,701; 5,955,256; 5,981,163;6,030,767; 6,087,141; and 6,106,773.

By using the preservative composition of the invention, the plateletscan be stored at room temperature. Platelet function also can be bettermaintained throughout the 5-day storage period mandated by the FDA.

The use of a hemoglobin-based oxygen carrier, even in small volumes, aspart of the preservative solution provides significantly greaterconcentration of oxygen than amounts currently made available by the useof oxygen-permeable storage bags. The combination of platelets with anoxygen carrier (e.g., a stroma-free hemoglobin solution) allows the useof gas impermeable bags, which reduces the high cost associated withusing gas permeable bags.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Tables are incorporated herein byreference.

The foregoing examples illustrate that an acellular preservativesolution for freshly collected platelets can be prepared for improvingthe functional half-life of platelets. The addition of the preservativesolution to freshly collected platelets better maintains the originalblood clotting function when infused during the storage period of theplatelets. The addition of a preservative solution permits an extendedstorage of the platelets at refrigeration temperatures, and allows theplatelets to maintain blood clotting properties without affecting thehalf-life of the platelets in circulation once transfused. As a result,the platelets stored for an extended period can be used for transfusionswhile saving a substantial amount of effort and cost.

EXAMPLE 1 General Procedure of Preparing the Preservative SolutionContaining Antiplatelet

In 50 ml of an acellular chemically modified hemoglobin-based carriersubstantially free of red cell membrane (stroma) contaminants, with ahemoglobin concentration of 12-20 gm/dl and a methemoglobinconcentration of less than 5%, the following active ingredients wereadded:

1) A GPIIb/IIIa inhibitor, such as eptifibatide (INTEGRILIN®,Schering-Plough Corporation, Kenilworth, N.J., U.S.A.), XJ757, DUP728,XR299 or aggrastat (tirofiban) in an amount of 0.001-5.0 mg.

2) An anti-inflammatory drug (NSAID), such as ibuprofen, in an amount of20-60 mg.

The above preservative solution can be added either to the bloodcollection bag containing the anticoagulant or to a separately attachedbag. If the platelets are going to undergo a sterilizing procedure,chemical or radiation, then the preservative composition is either addedprior to sterilization or added in a separately attached bag.

3) A short to ultra-short acting Xa inhibitor such as DX-9065a,BX-80783, RPR-120844 or an Xa inhibitor from the SEL-series or othershort acting Xa inhibitor in an amount of 0.001-5 mg.

4) An energy source such as glucose or citrate to sustain aerobicmetabolism

5) Electrolytes such as Na, Cl, and Mg.

TABLE 1 provides the concentration ranges for some commonly used energysources and electrolytes.

TABLE 1 Commonly Used Energy Sources and Electrolytes ComponentConcentration (mM) NaCl 80 to 120 KCl 5 to 15 MgCl₂/MgSO₄ 2 to 5 Na₂Citrate 5 to 40 NAH₂PO₄/Na₂ HPO₄ 5 to 30 Na Acetate 20 to 40 NaGluconate 15 to 30 Glucose 20 to 50 Maltose 25 to 35 D-Mannitol 25 to 40

EXAMPLE 2 In vitro Assessment of Platelet Function and Stability

Cell counts in the platelet concentrates and mean platelet volume weredetermined electronically using a particle counter. The pH, pO₂, pCO₂,and bicarbonate levels were determined in a blood gas analyzer. Glucose,lactic acid, and lactic dehydrogenase levels in the plateletconcentrates were measured by standard clinical chemistry methodology.Platelet function was measured by aggregometry using ADP and collagen asagonists and by thrombelastography (TEG).

Thrombelastography (TEG)

The principle of TEG is based on the measurement of the physicalviscoelastic characteristics of blood clot. Clot formation was monitoredat 37° C. in an oscillating plastic cylindrical cuvette (“cup”) and acoaxially suspended stationary piston (“pin”) with a 1 mm clearancebetween the surfaces, using a computerized Thrombelastograph (TEG Model3000, Haemoscope, Skokie, Ill.). The cup oscillates in either directionevery 4.5 seconds, with a 1 second mid-cycle stationary period;resulting in a frequency of 0.1 Hz and a maximal shear rate of 0.1 persecond. The pin is suspended by a torsion wire that acts as a torquetransducer. With clot formation, fibrin fibrils physically link the cupto the pin and the rotation of the cup as affected by theviscoelasticity of the clot (Transmitted to the pin) is displayedon-line using an IBM-compatible personal computer and customizedsoftware (Haemoscope Corp., Skokie, Ill.). The torque experienced by thepin (relative to the cup's oscillation) is plotted as a function of time(FIG. 1).

TEG assesses coagulation by measuring various parameters such as thetime latency for the initial initiation of the clot (R), the time toinitiation of a fixed clot firmness (k) of about 20 mm amplitude, thekinetic of clot development as measured by the angle (∥), and themaximum amplitude of the clot (MA). The parameter A measures the widthof the tracing at any point of the MA. Amplitude A in mm is a functionof clot strength or elasticity. The amplitude on the TEG tracing is ameasure of the rigidity of the clot; the peak strength or the shearelastic modulus attained by the clot, G, is a function of clot rigidityand can be calculated from the maximal amplitude (MA) of the TEGtracing.

The following parameters were measured from the TEG tracing (FIG. 1):

R, the reaction time (gelation time) represents the latent period beforethe establishment of a 3-dimensional fibrin gel network (with measurablerigidity of about 2 mm amplitude).

Maximum Amplitude (MA, in mm), is the peak rigidity manifested by theclot.

Shear elastic modulus or clot strength (G, dynes/cm2) is defined by:G=(5000 A)/(100-A).

Blood clot firmness is important function parameters for in vivothrombosis and hemostasis because the clot must stand the shear stressat the site of vascular injury. TEG can assess the efficacy of differentpharmacological interventions on various factors (coagulationactivation, thrombin generation, fibrin formation, platelet activation,platelet-fibrin interaction, and fibrin polymerization) involved in clotformation and retraction.

Blood Sampling

Blood was drawn from consenting volunteers under a protocol approved bythe Human Investigations Committee of William Beaumont Hospital. Usingthe two syringe method, samples were drawn through a 21 gauge butterflyneedle and the initial 3 ml blood was discarded. Whole blood (WB) wascollected into siliconized Vacutainer tubes (Becton Dickinson,Rutherford, N.J.) containing 3.8% trisodium citrate such that a ratio ofcitrate whole blood of 1:9 (v/v) was maintained. TEG was performedwithin 3 hrs of blood collection. Calcium was added back at a finalconcentration of 1-2.5 mM followed by the addition of the differentstimulus. Calcium chloride by itself at the concentration used showedonly a minimal effect on clot formation and clot strength.

Statistical Analysis

Data are expressed as mean±SEM. Data were analyzed by either paired orgroup analysis using Student's t-test or ANOVA when applicable;differences were considered significant at P<0.05 or less.

Effect of GPIIb/IIIa Antagonists c7E3 (Long Acting Versus Short/UltraShort Acting) On Tissue Factor Mediated Clot Retraction in Human WholeBlood Thrombelastography

Increasing concentrations of GPIIb/IIIa antagonists impaired the rate ofincrease in G force developed without prolonging R by tissue factor(TF)-activated whole blood clots. FIGS. 2A-2C are representativetracings showing efficacy of GPIIb/IIIa antagonist c7E3 in inhibitingTF-mediated clot formation under shear use of TEG.

Determination of Dissociation Rates

To determine platelet/GPIIb/IIIa ligand dissociation rate (t_(1/2)),platelet rich plasma samples were treated for 60 minutes with 0.04 μM of³H-Roxifiban or the various Roxifiban isoxazoline analogs includingXR299, DMP802, and XV454. Following this 60-minute incubation period,the tubes were centrifuged for 10 minutes (150×g). The resulting³H-radioligand/platelet rich plasma (PRP) complex was carefully removedand centrifuged for an additional 10 minutes (1,500×g). The resultingplatelet poor plasma (PPP) was removed and the platelet pelletre-suspended (1.6×10⁸/ml) in fresh PPP. Five hundred microliters of thissuspension was transferred to wells of a 24-well plate (blocked with 5%bovine serum albumine (BSA)). To initiate dissociation, dilution with1.0 ml Tris buffer, pH 7.4 containing 100 μM non-radiolabeled ligand wasadded to the wells. At designated time points (0-60 minutes), the³H-GPIIb/IIIa antagonist/PRP complex was removed from the wells. ForGPIIb/IIIa antagonists with fast platelet dissociation rate the t_(1/2)(min.) for the dissociation of platelet bound ³H-GPIIb/IIIa antagonistswas carried out at short intervals. The resulting platelet pellet wascounted using a liquid scintillation counter. CPMs recovered arecompared to the control (t=0) and presented as percent bound per 0.8×10⁸platelets over time. The t1/2 for the platelet dissociation of short toultra-short acting GP IIb/IIIa antagonists including Integrilin,Tirofiban, XR299, XV454, XV457 ranged from 0.05-0.25 minutes (Table 3).As used herein, the term “short-to-ultra-short” refers to the half lifeof the compound that is based on (i) in vitro displacement results asshown in Table 3 and is based on (ii) in vivo half life after theinfusion of the preserved platelets, which is less than 4 hours inhuman.

Radiolabel/Platelet Elution Profile

Preparation of Gel Columns and Platelets

About 200 ml of gel slurry (Sepharose-CL4B, #17-0150-01, Pharmacia) wasplaced in a large buchner funnel and washed with 1 liter of distilledwater. The washed gel was reconstitute with water to form a thickslurry. Two columns were prepared by loading about 120 ml of the washedgel slurry to each column (Siliconize column with Sigma-Cote, SigmaChemical #SL-2; or Bio-Rad Column #737-2531). One column was used todetermine platelet counts, while the other was used to determine CPMs.The packed columns were washed with 250 ml of distilled water, followedwith 150 ml of HBMT through the gel to allow buffer/gel equilibration.

Human whole blood is collected via venupuncture as described above andplaced in intovacutainers containing 0.5 ml of 0.1 M buffered sodiumcitrate (Becton/Dickinson). The whole blood was centrifuged for 10minutes at 150×g (˜1000 rpm Sorvall RT6000). The PRP was collected andkept in a capped polypropylene tube until use.

Radiolabeling and Platelet Elution

3.5 ml of PRP was incubated with either a radiolabeled ligand or a coldligand for 15 minutes at 22° C. The PRP was then carefully loaded onto aSepharose gel column and eluted with Hepes Buffered Modified TyrodesSolution (HBMT, see Table 2)) and collect 60, 2 ml fractions (totalvolume=˜120 ml) from each column.

Non radioactive fractions were counted on a coulter counter (T540), inorder to determine platelet counts. 250 μl of the radiolabeled elutionsfrom each fraction were counted on a beta counter (Packard MinaxiTri-Carb). The labeling/eluting process is summarized in FIG. 3

TABLE 2 Hepes Buffered Modified Tyrodes Solution (HBMT) CHEMICAL [M] g/lNaCl 0.14 8.2 KCl 0.0027 0.201 NaH₂PO₄ + H₂O 0.0004 0.055 NaHCO₃ 0.0121.0 GLUCOSE 0.0055 0.991 BSA fraction-V 3.5 HEPES 0.005 1.192 NOTE: pH7.4 at 22° C.

Table 3 shows dissociation rates of short acting (e.g., XR290) and longacting (e.g., c7E3) GPIIb/IIIa antagonists to human platelets

TABLE 3 Long acting Short to ultra-short acting Binding Parameters XV459XV454 c7E3 DMP728* XR290* Tirofiban* Integrilin* Dissociation Rates 7.032.0 40.0 0.2 3.05 0.1 0.25 (t½ - Minutes)

In vitro t1/2 of 0.1 minute translate to 30 minutes washout in vivo inanimals or human, t1/2 of 7 minutes translate into 20-24 hours washoutin vivo in animals or human, and t1/2 of 30-40 minutes translate towashout of 7-10 days (the life time of the platelet in the circulation).

* For platelet preservation, a short to ultra-short GPIIb/IIIaantagonists will be the preferred ones.

EXAMPLE 3 In vivo Assessment of Platelet Function and Stability Example3(a) Circulation Half-Life

The platelets stored in the preservative solution were radioactivelylabeled by C15 or In111. The labeled platelets were injected intorabbits via the ear vein. Blood samples were drawn at different timepoints and the radioactively measured. The radioactive decay curveprovided an estimate of the circulating half-life of the platelets. Theresulting decay curve was compared with traditionally stored plateletsthat were labeled in a similar manner.

Example 3(b) In vivo Bleeding Model

A rabbit model was made thrombocytopenic (platelet counts approximately30,000 per*L or 10% of normal) by subcutaneous injections of busulfan(25 mg/kg). Two weeks following busulfan treatment, the rabbits weretreated by the surgery procedures described by Krishnamurti et al,Transfusion, 39:967 (1999). Blood samples were obtained at differenttime points and the survival of the platelets determined. These valueswere compared to that obtained by traditionally stored platelets.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence whichis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

1. A platelet preservation package, comprising: a platelet preservationcomposition comprising: a short-to-ultra-short acting antiplateletagent; a short-to-ultra-short acting anticoagulant; an oxygen carrierselected from the group consisting of hemoglobin, ferroprotoporphyrin,perfluorochemicals, and mixtures thereof; and a container for storingthe platelet preservation composition.
 2. The platelet preservationpackage of claim 1, wherein said antiplatelet agent is capable ofreversibly blocking platelet activation and/or aggregation by blockingsites on the platelet surface.
 3. The platelet preservation package ofclaim 1, wherein said antiplatelet agent is capable of binding to, orassociating with a GPIIb/IIIa receptor site.
 4. The plateletpreservation package of claim 1, wherein said antiplatelet agent isselected from the group consisting of DUP728, XJ757, XR299,eptifibatide, orbofiban, xemilofiban, lamifiban, tirofiban, and mixturesthereof.
 5. The platelet preservation package of claim 1, wherein saidantiplatelet agent is selected from the group consisting of linear andcyclic RGD-containing peptides.
 6. The platelet preservation package ofclaim 5, wherein said linear and cyclic RGD-containing peptides areconjugated to a Nitric oxide donor.
 7. The platelet preservation packageof claim 1, wherein said antiplatelet agent is a pharmaceuticallyacceptable antiplatelet agent and has a circulating half-life ofinhibition of about less than 4 hours.
 8. The platelet preservationpackage of claim 1, wherein said antiplatelet agent comprises about0.001 mg to about 5 mg of a GPIIb/IIIa inhibitor.
 9. The plateletpreservation package of claim 1, wherein said anticoagulant is acompound that reversibly inhibits factor Xa, or factor IIa, or both. 10.The platelet preservation package of claim 9, wherein said anticoagulantis a short-to-ultra-short acting IIa inhibitor, and wherein saidshort-to-ultra-short acting IIa inhibitor is selected from the groupconsisting of hirudin, hirulog, melgatran, and combinations thereof. 11.The platelet preservation package of claim 9, wherein said anticoagulantis a pharmaceutically acceptable anticoagulant and is a combination ofat least one short-to-ultra-short acting IIa inhibitor, and at least oneshort-to-ultra-short acting Xa inhibitor.
 12. The platelet preservationpackage of claim 9, wherein said anticoagulant is a short-to-ultra-shortacting Xa inhibitor with a circulating half-life of less than 4 hours.13. The platelet preservation package of claim 12, wherein saidanticoagulant is selected from the group consisting of DX-9065a,BX-80783, RPR-120844, and short-to-ultrashort acting Xa inhibitors. 14.The platelet preservation package of claim 1, wherein said antiplateletagent is a compound that binds to or associates with GPIIb/IIIa sites,and wherein the anticoagulant is a factor Xa inhibitor, a factor IIainhibitor, or a combination of both.
 15. The platelet preservationpackage of claim 1, wherein said oxygen carrier is a hemoglobin-basedoxygen carrier.
 16. The platelet preservation package of claim 15,wherein said hemoglobin-based oxygen carrier is substantially free ofred cell membrane contaminants.
 17. The platelet preservation package ofclaim 1, wherein the preserved platelet composition further comprises ashort-to-ultrashort acting broad spectrum anti-microbial agent selectedfrom the group consisting of: penicillin, monobactam, cephalosporin,carbapenems, vancomycin, isoniazid, ethambutol, aminoglycoside,tetracycline, chloramphenicol, macrolide, rifamycin, quinolone,fluoroquinolone, sulfonamide, polyene antibiotic, imidazole, triazole,griseofulvin and combinations thereof.
 18. The platelet preservationpackage of claim 1, wherein the container is an oxygen-permeable bag.19. The platelet preservation package of claim 1, wherein the containeris an oxygen-impermeable bag.